Transgenic production of antibodies in milk

ABSTRACT

A method for the production of monoclonal antibodies in mammal&#39;s milk, through the creation of transgenic animals that selectively express foreign antibody genes in mammary epithelial cells.

FIELD OF THE INVENTION

This invention pertains to a method for the production of monoclonalantibodies in mammal's milk, specifically through the creation oftransgenic animals that selectively express foreign antibody genes inmammary epithelial cells.

BACKGROUND OF THE INVENTION

Immunoglobulins are heteropolymeric proteins that are normallysynthesized, modified, assembled, and secreted from circulating Blymphocytes. Using recombinant DNA technology, it is possible to programcells other than B-lymphocytes to express immunoglobulin genes Thedifficulties encountered in this effort stem from several factors: 1)Both heavy and light chains of immunoglobulins must be co-expressed atappropriate levels; 2) Nascent immunoglobulin polypeptides undergo avariety of co- and post-translational modifications that may not occurwith sufficient fidelity or efficiency in heterologous cells; 3)Immunoglobulins require accessory chaperone proteins for their assembly;4) The synthetic and secretory capacity of the cell may be inadequate tosecrete large amounts of heterologous proteins; and 5) The secretedimmunoglobulins may be unstable in the extracellular milieu of a foreigncell.

Because immunoglobulins have many therapeutic, diagnostic and industrialapplications, there is a need in the art for expression systems in whichthese proteins can be reproducibly manufactured at a high level, in afunctional configuration, and in a form that allows them to be easilyharvested and purified. The development of transgenic animal technologyhas raised the possibility of using large animals as geneticallyprogrammed protein factories. P.C.T. application WO 90/04036 (publishedApr. 19, 1990) discloses the use of transgenic technology forimmunoglobulin expression. WO 92/03918 (Mar. 19, 1992) and WO 93/12227(Jun. 24, 1993) teach the introduction of unrearranged immunoglobulingenes into the germline of transgenic animals. The use of intactimmunoglobulin genes (including their respective promoter regions) willresult in their expression in lymphocytes and secretion into thebloodstream of the host animal; this necessitates a strategy forsuppressing the expression of the host's endogenous immunoglobulins, andraises the problem of purifying the immunoglobulins from serum, whichcontains many other proteins, including proteolytic enzymes.Furthermore, if the transgenic approach is chosen, heavy and light chaingenes must both be incorporated into the host genome, in a manner thatenables their comcomittant expression.

Another option in creating transgenic animals is to link the gene ofinterest to a heterologous transcriptional promoter that only functionsin a defined cell type within the host. In this manner, tissue-specificexpression of the transgene may be programmed. U.S. Pat. No. 4,873,316(issued Oct. 10, 1989) discloses the production of recombinant tissueplasminogen activator (TPA) in the milk of transgenic mice in which theTPA gene is linked to the promoter of the milk protein casein. Otherproteins that have been expressed in a similar fashion include cysticfibrosis transmembrane conductance regulator (DiTullio et al.,Bio/Technology 10:74, 1992), urokinase (Meade et al., Bio/Technology 8:443, 1990), interleukin-2 (Buhler et al., Bio/Technology 8:140, 1990),and antihemophilic factor IX (Clark et al., Bio/Technology 7:487, 1989).Notably, these proteins are all simple single-chain polypeptides that donot require multimerization or assembly prior to secretion.

It has now been found that when a transgenic mammal is created carryingpaired immunoglobulin light and heavy chain genes under the control ofthe casein promoter, such an animal produces large amounts of assembledimmunoglobulins which are secreted in its milk. Using the DNA constructsof the present invention, a surprisingly high efficiency ofco-integration of heavy and light chain genes is observed. Using themethod and constructs of the present invention, it is possible for thefirst time to program a mammary epithelial cell to produce and assemblecomplex tetrameric glycoproteins and secrete them in high quantities.

Accordingly, it is an object of the present invention to provide methodsfor the large-scale production of immunoglobulins in the milk oftransgenic mammals.

Another object of the invention is to provide methods for the design ofsynthetic immunoglobulins that can be produced in large quantities inmilk.

Yet another object of the invention is to provide methods foradministering therapeutically beneficial antibodies to suckling young,by creating female mammals that secrete such antibodies into their milk.

A further object of the invention is a transgenic non-human mammalhaving germ and somatic cells with recombinant DNA sequences encodingimmunoglobulin light and heavy chains, where said sequences areoperatively linked at their 5' termini to a mammary specific promoterand at their 3' end to a sequence comprising a polyadenylation site.

A further object of the invention is a casein promoter cassettecomprising in the 5' to 3' direction:

a) 5' promoter sequences from the beta casein gene,

b) an XhoI restriction site, and

c) 3' untranslated sequences from the goat beta casein gene.

These and other objects of the present invention will be apparent tothose of ordinary skill in the art in light of the presentspecification, drawings, and claims

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the Bc62 plasmid, which containsa 13.9 kb Sal I fragment that comprises cDNA encoding immunoglobulinlight chain, flanked on its 5' and 3' termini by goat beta caseinsequences.

FIG. 2 is a schematic representation of the Bc61 plasmid, which containsa 14.6 kb Sal I fragment that comprises cDNA encoding immunoglobulinheavy chain, flanked on its 5' and 3' termini by goat beta caseinsequences.

FIG. 3 depicts the immunoblot detection of human immunoglobulin heavychain in the milk of transgenic mice that were created using the betacasein promoter-linked immunoglobulin genes shown in FIGS. 1 and 2.

FIG. 4 depicts the immunoblot detection of human immunoglobulin lightchain in the milk of transgenic mice that were created using the betacasein promoter-linked immunoglobulin genes shown in FIGS. 1 and 2.

SUMMARY OF THE INVENTION

In one aspect, this invention comprises a method for obtainingheterologous immunoglobulins from the milk of transgenic mammals.Another aspect of the prevent invention comprises the method forcreating transgenic mammals by introducing into their germlineimmunoglobulin cDNA linked to a milk-specific promoter.

In another aspect, the present invention comprises transgenic mammalshaving germ cells and somatic cells having recombinant DNA sequencescomprising immunoglobulin cDNA linked to a milk-specific promoter.

In still another aspect, the present invention comprises an isolated DNAcomprising an expression cassette having 5' and 3' non-coding sequencesderived from the goat beta casein gene linked via a unique restrictionsite that serves as a convenient cloning site for immunoglobulin codingsequences.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature cited in thisspecification are hereby incorporated by reference in their entirety. Inthe case of inconsistencies, the present disclosure will prevail.

The present invention pertains to a method for the production ofmonoclonal antibodies that are secreted into the milk of transgenicanimals and the method for production of such animals. This is achievedby engineering DNA constructs in which DNA segments encoding specificpaired immunoglobulin heavy and light chains are cloned downstream of apromoter sequence that is preferentially expressed in mammary epithelialcells. The recombinant DNAs containing the promoter-linked heavy andlight chain genes are then coinjected into preimplantation embryos. Theprogeny are screened for the presence of both transgenes. Representativefemales from these lines are then milked, and the milk is analyzed forthe presence of the monoclonal antibody. In order for the antibody to bepresents both heavy and light chain genes must be expressed concurrentlyin the same cell. The antibodies may be purified from the milk, or themilk itself, comprising the immunoglobulins, may be used to deliver theantibodies to a recipient. This is discussed below.

The immunoglobulin genes useful in the present invention may be obtainedfrom natural sources e.g. individual B cell clones or hybridomas derivedtherefrom. Alternately, they may comprise synthetic single-chainantibodies in which the light and heavy variable regions are expressedas part of a single polypeptide. Furthermore, recombinant antibody genesmay be used that have been predictively altered by nucleotidesubstitutions that do or do not change the amino acid sequence, byaddition or deletion of sequences, or by creation of hybrid genes inwhich different regions of the polypeptide are derived from differentsources. Antibody genes by their nature are extremely diverse, and thusnaturally tolerate a great deal of variation. It will be appreciated bythose skilled in the art that the only limitation for producing anantibody by the method of the present invention is that it must assembleinto a functional configuration and be secreted in a stable form intothe milk.

The transcriptional promoters useful in practicing the present inventionare those promoters that are preferentially activated in mammaryepithelial cells, including promoters that control the genes encodingmilk proteins such as caseins, beta lactoglobulin (Clark et al., (1989)Bio/Technology 7: 487-492), whey acid protein (Gordon et al., (1987)Bio/Technology 5: 1183-1187), and lactalbumin (Soulier et al., (1992)FEBS Letts. 297: 13). Casein promoters may be derived from the alpha,beta, or kappa casein genes of any mammalian species; a preferredpromoter is derived from the goat beta casein gene (DiTullio, (1992)Bio/Technology 10:74-77).

For use in the present invention, a unique XhoI restriction site isintroduced at the 3' terminus of the promoter sequence to allow theroutine insertion of immunoglobulin coding sequences. Preferably, theinserted immunoglobulin gene is flanked on its 3' side by cognategenomic sequences from a mammary-specific gene, to provide apolyadenylation site and transcript-stabilizing sequences. Transcriptionof the construct in vivo results in the production of a stable mRNAcontaining casein-derived 5' untranslated sequences upstream of thetranslational initiator codon of the immunoglobulin gene and 3'untranslated sequences downstream of the translational termination codonof the immunoglobulin gene. Finally, the entire cassette (i.e.promoter-immunoglobulin-3 ' region) is flanked by restriction sites thatenable the promoter-cDNA cassette to be easily excised as a singlefragment. This facilitates the removal of unwanted prokaryoticvector-derived DNA sequences prior to injection into fertilized eggs.

The promoter-linked immunoglobulin heavy and light chain DNAs are thenintroduced into the germ line of a mammal e.g. cow, sheep, goat, mouse,oxen, camel or pig. Mammals are defined herein as all animals, excludinghumans, that have mammary glands and produce milk. Mammalian speciesthat produce milk in large amounts over long periods of time arepreferred. Typically, the DNA is injected into the pronuclei offertilized eggs, which are then implanted into the uterus of a recipientfemale and allowed to gestate. After birth, the putative transgenicanimals are tested for the presence of the introduced DNA. This iseasily achieved by Southern blot hybridization of DNA extracted fromblood cells or other available tissue, using as a probe a segment of theinjected gene that shows no cross hybridization with the DNA of therecipient species. Progeny that show evidence of at least one copy ofboth heavy and light-chain immunoglobulin genes are selected for furtheranalysis.

Transgenic females may be tested for immunoglobulin secretion into milk,using any of the immunological techniques that are standard in the art(e.g. Western blot, radioimmunoassay, ELISA). The anti-immunoglobulinantibodies used in this analysis may be polyclonal or monoclonalantibodies that detect isolated heavy or light chains or others thatreact only with fully assembled (H2L2) immunoglobulins.

The recombinant immunoglobulins are also characterized with respect totheir functionality, i.e. binding specificity and affinity for aparticular antigen. This is achieved using immunological methods thatare standard in the art, such as Scatchard analysis, binding toimmobilized antigen, etc. The stability characteristics of animmunoglobulin in the milk of a given species are also assayed, byapplying the above-described detection methods to milk that has beenincubated for increasing times after recovery from the animal.

The immunoglobulins produced by the methods of the present invention maybe purified from milk, using adsorption to immobilized Protein G. columnchromatography, and other methods known to those of ordinary skill inthe art of antibody purification.

The level of production of recombinant immunoglobulins in an individualtransgenic mammal is primarily determined by the site and manner ofintegration of the transgene after injection into the fertilized egg.Thus, transgenic progeny derived from different injected eggs may varywith respect to this parameter. The amount of recombinant immunoglobulinin milk is therefore monitored in representative progeny, and thehighest-producing females are preferred.

Those skilled in the art will recognize that the methods of the presentinvention can be used to optimize the production of natural andsynthetic immunoglobulins. The steps of creating a transgenic animal,testing for the presence of both heavy and light-chain genes, assayingthe secretion of immunoglobulin into the milk of female progeny, and,finally, assessing the quality of the resulting antibodies, can berepeated sequentially, without undue experimentation, to establishpreferred constructs for different applications.

According to the present invention, the nature of the recombinantimmunoglobulins and their specific mode of use can vary. In oneembodiment, the present invention encompasses high-level expression ofantibodies that are harvested and purified from milk and used inpurified form. High-level expression is defined herein as the productionof about 1 mg/ml of protein. In another embodiment, antibodies areengineered that provide protection to humans against infectiousdiseases; therapeutic administration is then achieved by drinking themilk. In a still further embodiment, lactating animals are engineered toproduce antibodies specifically beneficial to their offspring, whichacquire them through suckling. In a still further embodiment, animalsproduce an antibody that protects the lactating mammal itself againstbreast pathogens e.g. bacteria that produce mastitis.

The unexpectedly high-volume expression of immunoglobulins using themethod and constructs of the present invention also allows the use ofsuch immunoglobulins in pharmaceutical and chemical settings. By way ofnon-limiting example the method of the present invention can be used toproduce high levels of tetrameric antibodies directed against variouspathogens (e.g. E. coli, Salmonella, hepatitis B virus), biologicallyactive peptides (e.g. erythropoietin, tissue plasminogen activator,gamma interferon) and for use in chemical reactions directed againstvarious enzymes. Monoclonal antibodies that bind to the transition stateof a chemical reaction can be used in industrial-scale production.Furthermore, monoclonal antibodies are often immobilized on columns foruse in the purification of biopharmaceuticals; in such cases, productionof the antibodies represents a significant fraction of the cost ofpurification. The methods of the present invention facilitate theproduction of high-volume, low cost antibody stocks for use in thesetypes of applications.

The present invention is further described in the following workingexamples, which are intended to illustrate the invention withoutlimiting its scope.

EXAMPLE 1

CONSTRUCTION OF A MILK-SPECIFIC PROMOTER CASSETTE

The present invention encompasses a recipient vector into which manydifferent immunoglobulin genes can be interchangeably inserted. Thevector contains 5' milk-specific promoter sequences and 3' untranslatedgenomic sequences that flank an XhoI cloning site. This cloning isunique because it is the only one present in the vector. Preferably, theentire expression cassette should be flanked by restriction sites thatallow the easy excision of the promoter-linked immunoglobulin gene.

In this Example, the promoter and 3' genomic sequences were derived fromthe goat beta casein gene. The gene was cloned and characterized asdescribed by Roberts et al., 1992, Gene 121:255-262, which is herebyincorporated by reference.

The expression cassette, prior to insertion of immunoglobulin genes,consists of 6.2 kb upstream of the translational start of the betacasein coding sequence and 7.1 kb of genomic sequence downstream of thetranslational stop of the beta casein gene. The TaqI site just upstreamof the translational start codon was changed to an XhoI site. Thisunique XhoI cloning site is at the junction of the upstream anddownstream sequences. It is this XhoI site, included in the sequenceCGCGGATCCTCGAGGACC, (SEQ. ID. NO. 1), into which recombinantimmunoglobulin genes are inserted. (D. Tullio, (1992) Bio/Technology10:74-77)

The 3' beta casein region begins at the PpuMI site found in Exon 7 andcontinues for 7.1 kb downstream. Included in this sequence are theremaining 18 bp of Exon 7, and all of Exon 8 and Exon 9. These encodethe 3' untranslated regions of the goat beta casein gene, and terminatewith the sequence:TAAGGTCCACAGACCGAGACCCACTCACTAGGCAACTGGTCCGTCCAGCTGTTAAGTGA SEQ. ID. NO.2.

To engineer restriction sites flanking the casein cassette, the goatbeta casein control sequences were first cloned into the SuperCoslvector (#251301, Stratagene, La JollA, Calif.) with flanking NotI andSalI sites. This plasmid was then modified by changing the NotI site toa SalI site. This created a 13.3 kb SalI fragment containing the betacasein expression cassette within the gbc163 vector.

Example 2

Construction of Promoter-linked Monoclonal Antibody Genes

In this Example, the genes encoding a human monoclonal antibody directedagainst a colon cancer cell-surface marker were linked to the caseinpromoter. cDNAs encoding the light and heavy chains of this antibodywere cloned from an antibody-secreting hybridoma cell line into apUC19-derived vector. The light and heavy chain cDNAs were present onHindIII/EcoRI fragments of 702 bp and 1416 bp, respectively.

To adapt the genes for insertion into the casein promoter cassette, XhoIrestriction sites were engineered at both ends of each DNA segment asdetailed below. In the same step, the region upstream of theimmunoglobulin translation initiation codon was modified so that itcontained sequences similar to those in the analogous region of the betacasein gene.

Light chain gene: The pUC19 plasmid containing the light chain cDNAinsert was digested with HindIII, blunt-ended by treatment with theKlenow fragment of DNA Polymerase I, and ligated to an oligonucleotidecontaining an XhoI recognition sequence (#1030, New England Biolabs,Beverly, Mass.).

The region immediately upstream of the initiating ATG was thenmutagenized using an oligonucleotide with the following sequence: 5' AGTGAA TTC ATG CTC GAG AGC CAT GGC CTG GATC 3' SEQ. ID. NO. 3. Digestion ofthe final plasmid with XhoI produced the modified light chain cDNA thatwas flanked by Xhol cohesive ends.

The light chain cDNA was then inserted into the unique XhoI cloning siteof the gbc163 expression vector described in Example 1, yielding plasmidBc62 (FIG. 1).

Heavy chain gene: The pUC19 plasmid containing the heavy chain cDNA wasmutagenized using an oligonucleotide with the following sequence: 5' AGTGAA TTC ATG CTC GAG AGC CAT GAA GCA CCTG 3' SEQ. ID. NO. 4. Theresulting plasmid contains an XhoI site upstream of the heavy chaintranslation initiation codon.

The downstream HindIII site was converted to an Xhol site using asynthetic adapter with the sequence 5' AGC TCC TCG AGG CC 3' SEQ. ID.NO. 5. Digestion of the modified plasmid with XhoI produced the the 1.4kb modified heavy chain cDNA flanked by XhoI cohesive ends. Thisfragment was then inserted into the unique XhoI cloning site of gbc163to yield Bc61 (FIG. 2).

Prior to injection, promoter-linked light and heavy chain genes wereisolated from Bc61 and Bc62, respectively, by digestion with SalI. Thefragments were then purified by gel electrophoresis followed by CsClequilibrium gradient centrifugation. The DNA was dialyzed extensivelyagainst distilled water prior to quantitation. Example 3

Production of Transgenic Mice

The casein promoter-linked DNA fragments encoding the immunoglobulinheavy and light chains, obtained as described in Example 2, wereinjected into fertilized mouse eggs using procedures that are standardin the art, as described in Hogan, B., Constantini, F., and Lacey, E.,Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring HarborLaboratories, 1986). The resulting progeny were then analyzed for thepresence of both antibody gene sequences. DNA was extracted from tailbiopsy material and probed using Southern blot analysis. The probes usedin the hybridization were the original cDNAs encoding the heavy andlight chains. As seen in Table 1, most of the first generationtransgenic progeny had incorporated both transgenes.

                  TABLE 1                                                         ______________________________________                                        Summary of Bc61 - Bc62 Mice                                                   Founder Sex        Bc61     Bc62   Expression                                 ______________________________________                                        1-2     M          Pos.     Pos.                                              1-3     M          Pos.     Pos.   light chain only                           1-9     M          Pos.     Pos.                                              1-15    F          Neg.     Pos.   Low level                                                                     lambda chain                               1-16    F          Pos.     Neg.                                              1-19    F          Pos.     Pos.   N.D.                                       1-23    F          Pos.     Pos.   1-3 mg/ml                                  1-24    F          Pos.     Pos.   low level                                  1-25    M          Pos.     Neg.                                              1-39    M          Pos.     Pos.                                              1-13    F          Pos.     Pos.   N.D.                                       1-56    F          Pos.     Pos.   N.D.                                       1-64    M          Pos.     Pos.                                              2-76    F          Pos.     Pos.   1-3 mg/ml                                  2-82    F          Pos.     Pos.   1-3 mg/ml                                  1-72    M          Pos.     Pos.                                              2-92    F          Pos.     Pos.   0.2-0.5 mg/ml                              2-95    F          Pos.     Pos.   0.2-0.5 mg/ml                              ______________________________________                                         N.D. = not detected                                                      

EXAMPLE 4

Analysis of Recombinant Immunoglobulins in Milk

Samples of milk from the transgenic mice obtained as described inExample 3 were analyzed for the presence of the heterologousimmunoglobulin by Western blot. The heavy chain of the antibody wasdetected using a horseradish peroxide-linked polyclonal antibodydirected against human gamma heavy chain (Antibody #62-8420, Zymed,South San Francisco, Calif.) as shown in FIG. 3. The light chain wasdetected using antibodies to the human lambda light chain, (Antibody#05-4120, Zymed, South San Francisco, Calif.) shown in FIG. 4. In theseFigures, it can be seen that immunoreactive heavy and light chains canbe detected in the milk of several animals, but not in the negativecontrol animal CD- 1. Human immunoglobulin can be detected in milk fromfounder 1-23 and from the progeny of the 1-76 and 1-72 founders. Theseanimals are the second-generation females, 2-76, 2-82, 2-92, and 2-95.The levels of expression range between 0.2 mg/ml to over 1 mg/ml (Table1)

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Capra hircus                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: beta casein 5'                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CGCGGATCCTCGAGGACC18                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Capra hircus                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: beta casein 3                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TAAGGTCCACAGACCGAGACCCACTCACTAGGCAACTGGTCCGTCCAGCTGTTAAGTGA59                 (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Capra hircus                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: light chain 5'                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AGTGAATTCATGCTCGAGAGCCATGGCCTGGATC34                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Capra hircus                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Heavy chain 5'                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AGTGAATTCATGCTCGAGAGCCATGAAGCACCTG34                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Capra hircus                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: HEAVY CHAIN 3'                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AGCTCCTCGAGGCC14                                                              __________________________________________________________________________

What is claimed is:
 1. A high level expression method for providing aheterologous and assembled immunoglobulin, in the milk of a transgenicmammal comprising:obtaining milk from a transgenic mammal havingintroduced into its germline a heterologous immunoglobulinprotein-coding sequence operatively linked to a promoter sequence thatresults in the preferential expression of said protein-coding sequencein mammary gland epithelial cells, thereby providing said heterologousand assembled immunoglobulin in the milk of said mammal, wherein saidheterologous and assembled immunoglobulin is a functional configurationand is produced at level of at least about 1 mg/ml in the milk of saidmammal.
 2. The method of claim 1 wherein said mammal is selected fromthe group consisting of mice, sheep, and pigs.
 3. The method of claim 1wherein said promoter is selected from the group consisting of the betalactoglobulin promoter, whey acid protein promoter, and the lactalbuminpromoter.
 4. The method of claim 1 wherein said immunoglobulin comprisesheavy and light chains.
 5. The method of claim 1 wherein saidimmunoglobulin is of human origin.
 6. The method of claim 1 wherein saidimmunoglobulin is purified from the milk of said mammal.
 7. The methodof claim 1 wherein said promoter is the casein promoter.
 8. A high levelexpression method for providing a heterologous and assembledimmunoglobulin, in the milk of a transgenic goat comprising:obtainingmilk from a transgenic goat having introduced into its germline aheterologous immunoglobulin protein-coding sequence operatively linkedto a promoter sequence that results in the preferential expression ofsaid protein-coding sequence in mammary gland epithelial cells, therebyproviding said heterologous and assembled immunoglobulin in the milk ofsaid goat, wherein said heterologous and assembled immunoglobulin is afunctional configuration and is produced at levels of at least about 1mg/ml in the milk of said goat.
 9. The method of claim 8 wherein saidpromoter is selected from the group consisting of the beta lactoglobulinpromoter, whey acid protein promoter, and the lactalbumin promoter. 10.The method of claim 8 wherein said immunoglobulin comprises heavy andlight chains.
 11. The method of claim 8 wherein said immunoglobulin isof human origin.
 12. The method of claim 8 wherein said immunoglobulinis purified from the milk of said goat.
 13. The method of claim 8wherein said promoter is the casein promoter.